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Investigating lead species and bioavailability in contaminated soils: Coupling DGT technique with artificial gastrointestinal extraction and in vivo bioassay Zhaodong Liu, Hong-bo Li, Xu Fang, Hao Zhang, Lena Q. Ma, and Jun Luo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06918 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019
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Investigating lead species and bioavailability in contaminated soils:
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Coupling DGT technique with artificial gastrointestinal extraction
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and in vivo bioassay
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Zhao-Dong Liu1, Hong-Bo Li1, Xu Fang1, Hao Zhang2, Lena Q. Ma1,3, Jun Luo1,*
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Environment, Nanjing University, Nanjing 210023, People’s Republic of China
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2
State Key Laboratory of Pollution Control and Resource Reuse, School of the
Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, United
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Kingdom
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3
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USA
Soil and Water Science Department, University of Florida, Gainesville, FL 32611,
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* Corresponding authors, 0086–25–89680632,
[email protected] ACS Paragon Plus Environment
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ABSTRACT
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Although strong in vivo-in vitro correlations (IVIVCs) between relative
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bioavailability (RBA) and bioaccessibility of soil Pb were well reported, knowledge on
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the fractions of bioaccessible Pb in simulated gastrointestinal (GI) fluids that are
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available for absorption into the systemic circulation is limited. Here, Pb-RBA in 14
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Pb-contaminated soils were assessed using an in vivo mouse bioassay and compared to
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Pb bioaccessibility by the gastrointestinal phase of the UBM (Unified Bioaccessibility
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research group of Europe (BARGE) Method) in vitro assay with and without 0.45-μm
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filtration of GI fluid. Results showed good IVIVC between Pb-RBA and Pb
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bioaccessibility without filtration (r2 = 0.62), while Pb bioaccessibility with filtration
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provided a poor correlation with Pb-RBA (r2 = 0.16). This suggested that besides
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dissolved Pb ions, Pb-complexes formed in the UBM gastrointestinal fluid might also
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contribute to bioavailable Pb. To ascertain this, DGT (diffusive gradients in thin-films)
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devices which can measure both Pb2+ ions and labile inorganic and organic Pb-
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complexes were introduced to the UBM fluids to measure Pb DGT-bioaccessibility,
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which showed strong correlation to Pb-RBA (r2 = 0.71). With increasing diffusive gel
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thickness which could enhance release of Pb ions from Pb-complexes, Pb DGT-
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bioaccessibility increased by 3.4–5.7 times, while inclusion of dialysis membrane
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within DGT devices significantly decreased Pb DGT-bioaccessibility by inhibiting
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diffusion of Pb complexes to binding gel. These results confirmed the contribution of
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Pb-complexes to Pb bioavailability, providing new insights to Pb bioavailability.
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INTRODUCTION
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Lead (Pb) is toxic to humans and may lead to serious health problems.1,
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various exposure pathways, incidental ingestion of Pb-contaminated soils represents a
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major contributor. This exposure scenario is more relevant for children who are more
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susceptible to soil ingestion than adults via hand-to-mouth behavior. Therefore,
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exposure assessment of Pb-contaminated soils via soil ingestion is receiving increasing
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attention.3 However, to pose a risk, ingested contaminants must become bioavailable
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and be absorbed in the bloodstream. Therefore, measurement of bioavailable Pb
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fraction in contaminated soils that can be absorbed into the blood systemic circulation
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is essential for accurate exposure assessment.4, 5
2
Among
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Based on different animal models, studies have measured Pb relative bioavailability
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(RBA, relative to Pb acetate exposure), which is defined as the fraction of an ingested
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Pb dose that crosses the gastrointestinal epithelium from test soil compared to Pb dose
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from soluble Pb acetate.6-9 However, the high experimental cost and time-consuming
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operations as well as the ethical issues related with in vivo animal models made in vitro
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assays good alternatives to predict Pb-RBA in soils. These assays measure contaminant
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concentrations that are solubilized following simulated human gastrointestinal (GI)
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extraction and potentially available for absorption into the systemic circulation (termed
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as bioaccessibility).10 Until now, there have been several developed methods:
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Solubility/Bioavailability
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Gastrointestinal method (IVG),11 Physiologically Based Extraction Test (PBET),12
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Deutsches Institutfür Normung e.V. method (DIN),13 Unified Bioaccessibility research
Research
Consortium
method
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(SBRC),7
In
Vitro
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group of Europe (BARGE) Method (UBM),14 and the Simulator of the Human
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Intestinal Microbial Ecosystem (SHIME).15 It is well documented that Pb
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bioaccessibility using different in vitro methods correlated well with Pb-RBA based on
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in vivo experiments using animals, indicating that in vitro method is a powerful tool for
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predicting soil Pb-RBA.11, 16, 17
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Although studies have proved the ability of in vitro simulation methods in
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measuring soil Pb-RBA, the methods only take into account the bioaccessible fraction
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which is considered to represent the maximum amount of soil Pb available for
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transporting across the intestinal epithelium. They do not consider different Pb species
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in the GI fluid with different transportation abilities across the intestinal epithelium.18,19
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The amount of metal transported across the intestinal epithelium is strongly related to
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the metal speciation.20 Besides free metal ions, labile metal species that can dissociate
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and produce Pb2+ ions would be available for transporting across the intestinal
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epithelium.21, 22 Since Pb absorption mainly happens in the intestine,23 investigation on
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the Pb speciation in the intestinal fluid can be helpful to understand Pb bioavailability
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in soils and the associated risk. Based on Pb ion selective electrode (Pb-ISE) and
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voltammetry techniques (differential pulse anodic stripping voltammetry, DPASV),
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Oomen et al.,24 discovered that the major contributor to dissolved Pb in the GI fluid
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was Pb bile-related complexes, which dissociated and produced Pb ions and
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subsequently absorbed. But these methods are complicated and suffer from
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interferences.25, 26 Therefore, a simple and more accurate technique that can provide
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direct information on Pb speciation in the GI fluids is needed.
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Diffusive gradients in thin-films technique (DGT) is a holistic tool for estimating
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bioavailability of metals in soils.27-29 A standard DGT device contains a diffusive gel
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of controllable pore size and thickness and a binding gel with strong binding resin. An
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analyte can diffuse through the diffusive gel and be adsorbed by the binding gel. Metal
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uptake by DGT from soil could deplete the soil solution concentration, inducing
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resupply of the metal from the solid phase and metal complexes to soil solution,
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providing continuous supply of the metal to DGT, which has been investigated by
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studies in soils.30, 31 The absorption of bioaccessible Pb in the GI fluids is also a dynamic
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process. The combination of in vitro simulation with DGT technique may overcome
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the limitation of in vitro simulation methods without considering the transmembrane
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absorption of bioaccessible Pb in the GI fluids. The DGT technique has been used to
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investigate metal bioaccessibility in the UBM fluid.32, 33 However, DGT-measured Pb
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in gastrointestinal fluids, which was defined as the fraction of bioaccessible Pb in the
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GI fluids can crosses the gastrointestinal epithelium and becomes available for
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distribution to internal target tissues and organs, has not been validated against Pb
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bioavailability using in vivo data.
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To test the bioavailability of bioaccessible Pb in gastrointestinal fluids, this study
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used a combined method of in vitro GI extraction and DGT speciation technique to
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investigate Pb species in GI fluids available for absorption and to assess the advantages
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of the combined method in predicting Pb-RBA. The UBM in vitro method was selected
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to measure soil Pb bioaccessibility since it has been well correlated with Pb
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bioavailability based on animal models,34,
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condition in both human gastric and intestinal tracts.18, 32, 33, 35 Mice were selected as an
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animal model to measure Pb-RBA in contaminated soils.16, 36 The specific objectives
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of this study were: (1) to investigate the relationship between DGT-measured Pb
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bioaccessibility and Pb-RBA in 14 contaminated soils and validate the potential of DGT
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as a prediction tool of Pb bioavailability and (2) to gain insight into the Pb species and
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their contribution to the Pb bioavailability in the intestinal fluids.
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MATERIALS AND METHODS
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as well as it mimics the physiological
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Soil Preparation. Fourteen soil samples were collected from different
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contaminated sites in China (Table S1). All soil samples were air-dried at room
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temperature (25○C) and passed through a
